Synthesis of Silk Fibroin–Glycopolypeptide Conjugates and Their

Sep 11, 2012 - Silk fibroin (SF), the natural fibrous protein created by the Bombyx mori silk worm, is being increasingly explored as a biomaterial fo...
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Synthesis of Silk Fibroin−Glycopolypeptide Conjugates and Their Recognition with Lectin Soumen Das,† Debasis Pati,† Neha Tiwari,† Anuya Nisal,‡ and Sayam Sen Gupta*,† †

Chemical Engineering Division and ‡Polymer Science and Engineering Division, CReST, National Chemical Laboratory (CSIR), Dr. Homi Bhabha Road, Pune-411 008, India S Supporting Information *

ABSTRACT: Silk fibroin (SF), the natural fibrous protein created by the Bombyx mori silk worm, is being increasingly explored as a biomaterial for tissue engineering due to its excellent mechanical strength, high oxygen/water permeability, and biocompatibility. It is also well known that surface modification of SF with organic ligands such as the extracellular protein binding Arg-Gly-Asp (RGD) peptides help adhesion and proliferation of cells bettera key requirement for it to function as extracellular matrices. In this work, we have conjugated synthetic glycopolypeptides (GPs) that were synthesized by controlled ring-opening polymerization of α-manno-lys N-carboxyanhydrides (NCAs) onto SF by using Cu catalyzed click reaction to synthesize a new hybrid material (SF−GP), which we believe will have both the mechanical properties of native SF and the molecular recognition property of the carbohydrates in the GP. By controlling the amount of GP grafted onto SF, we have made three SF−GP conjugates that differ in their ability to assemble into films. SF−GP conjugates having a very high content of GP formed completely water-soluble brush-like polymer that displayed very high affinity toward the lectin concanavalin-A (Con-A). Films cast from SF−GP conjugates using lower amounts of grafted GP were more stable in water, and the stability can be modulated by varying the amount of GP grafted. The water-insoluble film SF−GP25 was also found to bind to fluorescently labeled Con-A, as was seen by confocal microscopy. Such SF−GP hybrid films may be useful as mimics of extracellular matrices for tissue engineering.

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INTRODUCTION Silk fibroin (SF), a natural fibrous protein created by the Bombyx mori silk worm, forms the silk fiber that has been extensively used in textile industry. SF is a high molecular weight biopolymer that is characterized by repeat hydrophobic and hydrophilic peptide sequences. The hydrophobic domains, consisting mainly of Gly-Ala-Gly-Ala-Gly-Ser repeats,1,2 constitute a large part of the silk proteins and leads to the formation of both inter- and intramolecular β-sheet structures3 that are responsible for the insolubility, high mechanical strength, and thermal stability of silk fibers. Aqueous solutions of SF have been reformed into gels, sponges, powder, and membranes and these have unique physiochemical properties.4 This versatility of the SF coupled with the excellent mechanical strength, high oxygen and water vapor permeability, biocompatibility, and biodegradability render them as a very interesting biomaterial for biomedical application such as tissue engineering and drug delivery.4−10 There have been several reports that have explored the use of SF-based scaffolds for tissue engineering.11−14 Although SF-based scaffolds have been shown to be excellent extracellular matrices for many cell types including fibroblasts, chondrocytes, and mesenchymal stem cells;15−19 the surface modification of such SF-based scaffolds by organic ligands have shown to improve the adhesive properties of these scaffolds toward cells.17,20−24 It is very well © 2012 American Chemical Society

understood that the interaction between cells and scaffolds (extracellular matrices) is vital in governing cell adhesion, proliferation, differentiation, migration, and sometimes even survival.25 This interaction is governed by a variety of factors characterizing the scaffold surface. For example, surface modification of SF-based scaffolds have been performed with peptides containing the Arg-Gly-Asp (RGD) motif since RGD binds very well to a large number of extracellular proteins, including collagen and fibronectin.20,26 This increases the adhesive property of the RGD-modified SF scaffolds and hence helps proliferate cells better. Similarly, carbohydrates are also well-known to recognize very specific receptors that are found on several cell surfaces. For example, the asialoglycoprotein receptor (ASGP-R) displayed on the hepatocyte cell surface interacts uniquely with galactose/N-acetyl-β-galactosamine containing carbohydrate ligands.27−30 This strategy has been used to design extracellular matrices bearing galactose residue for liver tissue engineering.30 It has been shown that hepatocytes adhere 8 times higher to polystyrene plates coated with lactose-conjugated SF than that coated with native SF.31,32 The interaction of carbohydrates with their corresponding Received: July 26, 2012 Revised: September 5, 2012 Published: September 11, 2012 3695

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Figure 1. Schematic representation for the formation of water-soluble brush-like polymer or water-insoluble film by varying the amount of GP grafted onto SF.

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receptor proteins are typically very weak, but their specificity can be increased several fold by displaying several copies of the carbohydrate on the ligand.33 Such multivalent interactions (polyvalency) have long been recognized for their potential to achieve effective adhesion between binding partners, even when individual, monovalent binding interactions are weak.34 Therefore, galactose containing synthetic glycopolymers can be used to guide hepatocyte adhesion through this unique ASGP-R− carbohydrate interaction and can act as an excellent ligand for the development of extracellular matrices for liver tissue engineering.30 In our group we have developed synthetic glycopolypeptides (GPs; glycopolymers with pendant carbohydrates on a polypeptide backbone) via the ring-opening polymerization (ROP) of glyco-N-carboxyanhydrides (glycoNCAs) by using very simple end functionalized primary amine initiators.35 Our methodology allows synthesis of such GPs with controlled molecular weight, glycosylation density, and positionattributes that are necessary for biological recognition processes.36 We have also shown that these polypeptides bind specifically and polyvalently to lectins.37 Since they mimic the composition of proteoglycans, they are expected to be biocompatible. We reasoned that if synthetic GPs can be covalently attached to SF, the new hybrid material will have both the mechanical properties of native SF and the molecular recognition property of the carbohydrates in the GP. This will give rise to a new class of functional material that we believe will be useful for several biological applications such as tissue engineering. In this report we describe a simple and efficient methodology to attach GPs to SF using click chemistry. Several SF-GP conjugates with varying GP-to-SF ratios were synthesized using our methodology. Depending upon the SF:GP ratio in the synthesized SF−GP conjugate, we were able to control the morphology of the SF−GP conjugates into either completely water-soluble brush-like polymers or water-insoluble films (Figure 1). The interaction of the SF−GP conjugates with the lectin concanavalin-A (Con-A) were studied in solution as well as with the films casted from SF−GP conjugates. Finally, we demonstrate preliminary experiments to show that the films formed from GP−silk conjugates were nontoxic to the rat skeletal muscle myoblasts L6 and could be used as a scaffold for adhesion and growth of these cells.

EXPERIMENTAL SECTION

Materials and Methods. Cocoons from Bombyx mori were obtained from Central Sericultural Research and Training Institute, Mysore. 4-Iodoaniline was obtained from Aldrich and converted to 4azidoaniline by using the literature procedure.38 Glyco-N-carboxyanhydride was prepared by using our previously published methodology.37 HAuCl4, triphosgene, propargyl amine, Con-A, and fluorescein isothiocyanate (FITC)-labeled Con-A was obtained from Aldrich. All the other chemicals used were obtained from Merck, India. Ultraviolet−visible (UV−vis) spectra were recorded on Carry-300 UV−vis spectrometer using 1 cm quartz cuvette at 25 °C, while Fourier transform infrared (FT-IR) spectra were recorded by using KBr pellets in a Perkin-Elmer FT-IR spectrum GX instrument. KBr pellets were prepared by mixing 97 mg of KBr with 3 mg of sample. 1H NMR spectra were recorded on Bruker spectrometers (200 MHz, 400 MHz). The NMR samples of SF, azido-SF, and SF−GP were prepared by dissolving freshly lyophilized samples in D2O at a concentration of 2 wt %. Preparation of Aqueous Silk Solution. The Bombyx mori silk cocoons were boiled in 0.5% (w/v) solution of Na2CO3 twice for about 30 min to remove the sericin coating. The resulting cottony mass of fibroin fibers was then dissolve in 9.3 M LiBr solution, keeping a concentration of 1gm/10 mL at 60 °C for 45 min. This solution was then dialyzed against distilled water for 48 h at 4 °C using a dialysis bag of cellulose acetate membrane with a molecular weight cutoff (MWCO) of 12 KDa. The water for the dialysis was changed at least six times: first after 3 h, then 6 h, and later 12 h each, and at last against borate buffer (100 mmol L−1 borate, pH 9.0) for an additional day. The solution was centrifuged at 12000 rpm for 20 min at 4 °C, and the supernatant was collected. The silk solution had a final concentration 3.5−4 wt %, which could be stored in a refrigerator for 3−4 months. Synthesis of Azido-SF. The diazonium coupling reaction on SF was performed according to the method reported by Kaplan and coworkers.17 4-Azidoaniline (33.5 mg, 0.25 mmol) was dissolved in 1.25 mL of acetonitrile and then mixed with aqueous solution of ptoluenesulfonic acid (0.625 mL, 1 mmol). This mixture was cooled on an ice bath followed by addition of aqueous solution of sodium nitrite (0.625 mL, 0.5 mmol). This reaction mixture was stirred for an additional 10 min to afford the diazonium salt in situ. This diazonium salt (0.5 mL; 0.05 mmol) was then added to the SF solution in borate buffer (2 mL, 4 wt %), and the reaction was allowed to proceed for 30 min at 0 °C. After completion of the reaction, the reaction mixture was purified using sephadex size-exclusion columns (NAP-25, GE Healthcare) that were pre-equilibrated with distilled water. Synthesis of GP. To a solution of α-manno-O-lys NCA (100 mg/ mL) in dry dioxane was added with a “proton sponge” 1,83696

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Recognition of the Mannose Residues in SF−GP by Lectin. Con-A interactions with SF−GP conjugates were studied in the 100 mM phosphate buffer solution (PBS; pH 7.2) containing 0.1 M NaCl, 0.1 mM CaCl2, and 0.1 mM MnCl2. Con-A was dissolved at 0.5 mg/ mL in this PBS buffer, and the resulting solution was mixed and sterile filtered. The solution was then diluted to 5 μM (based on Con-A tetramer at 104 000 Da). Turbidity measurements were performed by adding 120 μL of the diluted Con-A solution to a dry quartz microcuvette (150 μL volume, 1 cm path length). A solution of the GP poly(α-manno-O-lys)-functionalized silk-fibroins (SF−GP25, SF− GP50, and SF−GP125) in PBS buffer was then gradually added (6 μL at 0.25, 0.5, and 0.8 mg/mL respectively) to the Con-A solution. Upon addition, the solution was mixed vigorously for 60 s using a vortex mixer and then placed in the spectrometer, and absorbance data were recorded at 490 nm (Figure 5). Films of SF and SF−GP25 were cast on glass slides from their aqueous solutions. These glass slides were treated with methanol for 1 h and then dried for 24 h. A 0.5 mg/mL solution of FITC−Con-A was prepared by dissolving FITC-Con-A in 100 mM phosphate buffer (pH 7.2) containing 0.1 M NaCl, 0.1 mM CaCl2 and 0.1 mM MnCl2. The insoluble films of SF and SF−GP25 were incubated in this FITC−ConA solution at room temperature for 30 min. The films were washed with 100 mM phosphate buffer (containing 0.1 M NaCl, 0.1 mM MnCl2 and 0.1 mM CaCl2) three times. Attachment of FITC−Con-A on the surface of SF−GP25 was observed by a LSM 710 Carl Zeiss laser scanning confocal microscope (Figure 6). Cell Culture. L6 rat muscle myoblast cells were cultured in Dulbecco’s modified Eagle Medium (low glucose) with 10% fetal bovine serum (FBS). At 90% confluency, the cells were detached using trypsin and then seeded in to the well plates. An aliquot (100 μL) of aqueous solution of SF-GP25 (0.5% w/v) was coated over 12 mm coverslips and kept for drying in a laminar flow hood for 24 h followed by vacuum drying for another 6 h. The coverslips were then transferred into the 24 well plates and then treated with methanol for 2 h to make it water insoluble and also to make it sterile against bacterial growth. Methanol was then removed, and the coverslips were kept for drying in the laminar flow hood for 12 h. L6 cells with passage number 9 were then seeded into the 24 well plates containing the coated coverslips at a density of 30 000 cells in 1 mL of media containing FBS. The well plate was then kept in a humidified incubator having 5% CO2 at 37 °C for 24 h. The cell growth and shape was monitored using epifluorescence microscopy. Cell Viability Assay. Cell viability in the monolayer culture was performed using LIVE/DEAD assay experiment. For this purpose, a combination of acridine orange and propidium iodide was used. The staining solution was prepared by adding 10 μL of propidium iodide (7.5 mM solution in PBS) and 1 μL of acridine orange (0.67 mM solution in PBS) in 1 mL of cell culture media. The media was aspirated from the well plate after 24 h, and the LIVE/DEAD stain was then added to it and incubated for 15 min at 37 °C. The stain was then aspirated and washed twice with PBS prior to epifluorescence imaging (Ziess).

bis(dimethylamino)naphthalene (1.0 equiv to monomer, 1 M) as an additive and freshly distilled propargyl amine (0.5 M) as the initiator inside the glovebox. The progress of the polymerization was monitored by FT-IR spectroscopy by comparing with the intensity of the initial NCA’s anhydride stretching at 1785 cm−1 and 1858 cm−1 (Supporting Information (SI) Figure 1). The reaction was generally completed within 16 h. Then the solvent from reaction mixture was removed under reduced pressure. The resulting residue was redissolved in dichloromethane, and then the polymer precipitated out by addition of methanol. The precipitated polymer was collected by centrifugation and dried to get white solid GP. Polymer 1b. 1H NMR (400.13 MHz, CDCl3): δ 1.10−1.80(m, 6H), 1.92−2.18(m, 12H), 2.97−3.35(m, 2H), 3.70−3.90(amide H’s), 3.95− 4.35(m, 3H), 4.95−5.35(m, 2H), 5.35−5.50(m,1H), 5.50−5.75(m, 1H), 5.70−6.25(amide H’s). Deprotection Procedure of GP. Sodium methoxide (10 equiv) was added to the solution of acetyl-protected GP in methanol (10 mg/ mL) and stirred for 6−7 h at room temperature. Reaction was quenched by addition of Amberlite IR 120 hydrogen form up to neutral pH and filtereing the solution. The filtrate was dried under reduced pressure and redissolved in deionized water and then transferred into a dialysis tubing MWCO of 2 KDa. The compound was dialyzed against deionized water for 3 days, with water changes once every 2 h for the first day and then thrice per day. Then the dialyzed polymer was lyophilized to get white fluffy solid GP. Polymer 1c. 1H NMR (400.13 MHz, D2O): δ 1.35−2.12(m, 6H), 2.28(s, 1H), 3.00−3.25(m, 2H), 3.60−3.90(m, 5H), 3.95−4.45(m, 2H), 5.80−5.95(m, 1H). Synthesis of SF−GP Conjugates Using Copper-Catalyzed Azide−Alkyne Cycloaddition (CuAAC). SF−GP conjugates were synthesized by Cu (I)-catalyzed cycloaddition reaction of propargylterminated fully deprotected GP with azido-SF in various proportions (SF:GP = 1:25, 1:50, and 1:125) (Table 1). For example, to an

Table 1. Composition of Different SF−GP Conjugates before and after the Click Reaction composition of SF and GP before click

a

after click

SF−GP conjugates

azido-SF 1 wt% (wt/vol)

molar ratio SF:GP

% wt fraction of GP

% wt fraction of GPa

SF-GP25 SF-GP50 SF-GP125

1 mL 1 mL 1 mL

1:25 1:50 1:125

25 40 65

∼25 ∼36

Calculated from 1H NMR integration (SI Figure 5).

aqueous solution of azido-SF (1 wt %; 1 mL) was added GP (25 eq, 3.19 mg), CuSO4, 5H2O (25 equiv; 0.16 mg), and sodium ascorbate (50 equiv; 0.25 mg), and the reaction was allowed to proceed at room temperature under nitrogen atmosphere for 24 h. The progress of the reaction was monitored by the disappearance of azide stretch at 2117 cm−1 in FT-IR spectra (SI Figure 4). After completion of the reaction, reaction mixture was directly transferred into a dialysis tubing (MWCO of 12 KDa), and the reaction mixture was dialyzed against deionized water for 1 day and then against ethylenediaminetetraacetic acid (EDTA) solution for another day. Finally, the reaction mixture was dialyzed against deionized water for an additional 2 days to afford the SF−GP conjugate SF−GP25. Assembly of SF−GP Conjugates into Films. Solutions containing SF−GP25 and SF−GP50 (2 wt % in water) were cast onto a polystyrene surface and dried in a laminar flow hood for 24 h. The films were then placed in vacuum for another 24 h. To make them water insoluble, these films were treated with 70% methanol in water for 1 h and dried in vacuum overnight. Contact Angle Measurement. The aqueous solution of SF and SF−GP conjugates (1 wt %) were spread on glass slide and dried for 24 h. Films were treated with methanol for 1 h and then dried again. Water contact angle was measured by the sessile drop method using a Digidrop contact angle meter with an 8 μL water drop size. At least three different areas of each film were analyzed.

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RESULTS AND DISCUSSION Synthesis of Azido-SF. The attachment of alkyneterminated GPs (alkyne-GPs) onto SF requires the installation of azido groups onto SF.The azide moiety was introduced into SF using the methodology developed by Kaplan and coworkers.17 In short, the azido functional groups were introduced into the tyrosine residues of SF via diazonium coupling chemistry, as shown in Scheme 1. Since there are 280 tyrosine residues present in SF, introduction of the azide group in the tyrosine allows attachment of a large number of azide groups onto the SF chain. During the coupling of the in situ generated 4-azidobenzediazonium salt onto SF, the color of the reaction mixture changed from colorless to reddish brown due to the newly formed azobenzene chromophore on the tyrosine residues. This modified SF can be easily analyzed by UV−vis 3697

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characteristic azide stretch at 2117 cm−1 (SI Figure 4). The unreacted GP and Cu (II) was removed from SF−GP conjugates by performing extensive dialysis of the sample against EDTA and DI water. Inductively coupled plasma (ICP) analysis of all the SF−GP samples was performed, and no presence of residual Cu was found in all samples. Three different SF−GP conjugates having SF-to-GP ratios of 1:25, 1:50, and 1:125 by moles were synthesized by this method. These will be referred to as SF−GP25, SF−GP50, and SF− GP125, respectively (Table 1). To investigate the attachment of GP onto azido-SF, 1H NMR spectra of all the SF−GP conjugates were recorded. The spectrum of all SF−GP conjugates exhibited a new broad peak due to methylene and methine protons of the mannose residue of the GP’s at 3.60−3.85 ppm in addition to resonances characteristic of the SF (Figure 3). The anomeric proton of mannose residue was clearly observed at 5.82 ppm as were the other proton peaks that were characteristic of the GP. The presence of proton peaks characteristic of both GP and SF together with the FT-IR spectra, which shows decrease of the characteristic azide peak in the SF−GP conjugate, indicates the successful incorporation of GP into SF by click chemistry. The exact composition of SF−GP25 and SF−GP50 was determined by 1H NMR spectra, from the relative intensity of the peak at 1.36 ppm due to the β-methyl protons of alanine residue in azido SF with respect to the peak characteristic of methylenic and methine protons of the carbohydrate moiety of the GP at 3.5−4.5 ppm (SI Figure 5). For SF−GP125, we were unable to determine the exact composition from 1H NMR since intensity of the peaks from the GP moiety was very high and overlapped with the 1.36 ppm peak of the β-methyl protons of alanine residue in azido SF. FT-IR Analysis of SF−GP’s β-Sheet Structure. Regenerated SF is known to form water-insoluble films by exposure to methanol or by physical shear.40,41 Exposure to methanol induces formation of a high percentage of β-sheets, which leads to the formation of films. The physical properties such as elasticity and porosity can be modulated, which makes these materials very exciting for cellular interaction and migration. Therefore, we attempted to make silk films from the GPmodified SF solution. Silk films were casted from 2 wt % aqueous solution of SF-GP by slow evaporation of water. These cast films were annealed in 70% methanol/water solution to induce the formation of the β-sheets. Films formed from SFGP125 got rapidly solubilized in water. The films formed from SF-GP50 swelled in water over a period of 1 h (approximately between 1.5 and 2.0 times) and finally got solubilized into

Scheme 1. Diazonium Coupling Reaction with Tyrosine Residues in Silk

and 1H NMR. UV−vis spectrum shows a strong absorption band corresponding to the azobenzene chromophore at 353 nm with a shoulder at 403 nm (Figure 2a). In 1H NMR spectra, the upfield shifting and broadening of the tyrosine peaks in azido SF confirms covalent attachment of the azobenzene chromophore onto the tyrosine moiety (Figure 2b). The concentration of azo groups was evaluated from the absorbance at 353 nm using an extinction coefficient of 22 000 M−1cm−1.17 It was estimated that 51% of the tyrosine present was modified to the corresponding azide, which suggests that one azido-SF chain contained approximately 142 azide moieties. Incorporation of the azide moiety was further confirmed with FT-IR spectra, which displayed the appearance of a peak at 2117 cm−1 that is characteristic of an organoazide (SI Figure 4). Synthesis of GP. Alkyne-terminated GPs were synthesized by the ROP of per-O-acetylated-D-manno-L-lysine NCA using propargylamine as the initiator. Using our methodology,37 alkyne-terminated GP based on a poly-L-lysine backbone with pendant sugar α-D-mannose was synthesized. Polymerization of α-D-manno-L-lysine NCA was carried out in the presence of a 1.0 equiv proton sponge using freshly distilled propargylamine as the initiator (M/I = 15) in dry dioxane (Scheme 2). The completion of the reaction was confirmed by the complete disappearance of the anhydride stretch of NCA at 1785 cm−1 and 1858 cm−1 in FT-IR spectra. The resulting polymer shows monomodal and narrow molecular distribution in GPC chromatogram. The acetyl protecting groups of the pendant sugar were completely removed by NaOMe in methanol. The complete removal of acetyl groups was confirmed by the absence of acetyl protons in the 1H NMR spectra of the watersoluble GP. Synthesis and Characterization of SF−GP Conjugates. The alkyne-terminated GP was attached with azido-SF by using the CuAAC reaction.39 The alkyne-terminated GP was coupled to the azide moiety of azido-SF to form a 1,2,3-triazole linkage between SF and GP using CuSO4,5H2O/sodium ascorbate as the catalyst (Scheme 3). The progress of the reaction was followed by FT-IR spectra by monitoring the decrease in the

Figure 2. (a) UV−vis spectra of azido-SF (red) and SF (black) in water at pH 7.0 showing the incorporation of azo-benzene. (b) 1H NMR spectra of azido-SF shows upfield shift and broadening of aromatic protons. 3698

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Scheme 2. Synthesis of Completely Water-Soluble GP with Terminal Alkyne Group

Scheme 3. Synthesis of SF−GP Conjugates via CuAAC

Figure 3. 1H NMR spectra SF−GP25 in D2O showing the peaks corresponding to GP in addition to SF peaks.

water over a period of 6 to 8 h. For SF-GP25, the films formed were completely insoluble in water and stable for weeks. Changes in the secondary structure of the SF−GP films induced by methanol treatment were studied by FT-IR spectroscopy.(Figure 4, Table 2) FT-IR of films formed from unmodified SF before and after methanol treatment was recorded for comparison (Table 2). The FT-IR spectra the SF before its methanol treatment exhibits absorption bands at amide I (1650 cm−1; carbonyl stretching), amide II (1540 cm−1; N−H bend), and amide III (1230 cm−1; C−N stretching) that are characteristic of the protein in the random coil conformation (Figure 4, Plot A). However, in methanol treated SF films, additional peaks at 1630 cm−1, 1520 cm−1 and 1270 cm−1 characteristic of β-sheets were observed, indicating that methanol treatment had induced the formation of β-

sheets17,40 (Figure 4, Plot B). Although the FT-IR spectra of the α-helical GP display absorption bands of amide I at 1655 cm−1, amide II at 1544 cm−1, and amide III at 1258 cm−1 (SI Figure 3), they do not overlap with the absorption bands in the SF films that are due to the formation of the β-sheets, which is critical for the stability of the film in water. Therefore, changes in the absorption bands characteristic of the β-sheets in the films formed from SF-GP conjugates can be used as a fingerprint to understand the effect of the conjugated GP on film formation. It was observed that with increasing GP content in SF-GP conjugates, the FT-IR absorption bands at 1630 cm−1, 1520 cm−1 and 1270 cm−1 that are characteristic of βsheets decreases. In SF-GP125, where the wt% of GP is expected to be higher than 50%, no bands characteristic of β-sheets were observed (Figure 4, Plot E). Therefore, high grafting of GP 3699

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Table 3. Water Contact Angle Values films

contact angle (o)

unmodified SF SF−GP25 SF−GP50

67.3 ± 3 44.7 ± 2 31.2 ± 4

GP content. Hence our methodology allows tuning of the hydrophilicity of the SF−GP conjugate films by changing the grafting density of GP on the SF. Recognition of the SF−GP Conjugates by the Lectin Con-A. The tetrameric protein Con-A is known to specifically bind to mannosyl and glucosyl residues. Hence the multivalent GP poly(α-manno-O-lys) functionalized silk-fibroins (SF−GP conjugates) are expected to interact with the tetrameric Con-A. In SF−GP conjugates, multiple copies of the mannosylated GPs are displayed onto the SF backbone, and they are expected to interact with the carbohydrate binding site on the tetrameric Con-A. Therefore, the recognition and binding affinity of SF− GP conjugates to Con-A were estimated by turbidity measurements.42 The turbidity variation having three different poly(α-manno-O-lys) functionalized silk-fibroin (SF−GP25, SF−GP50, and SF−GP125) with Con-A is presented in Figure 5. As can be observed for all the polymers, the turbidity first

Figure 4. IR spectra of three SF−GP conjugate films and unmodified SF film before and after of its methanol treatment.

onto SF inhibits formation of the β-sheets since the presence of high amounts of GP does not allow crystallization of the hydrophobic repeat units in SF. Hence, the films formed from SF−GP125 are water-soluble, and the SF−GP125 conjugate probably has a brush-like structure in solution. By contrast, the presence of β-sheets is observed in films formed from both SF− GP50 and SF−GP25, although the amide III peak at 1270 cm−1 qualitatively suggests that the content of β-sheets is higher in SF−GP25 films than in the corresponding SF-GP50 films. This is also expected since higher amounts of GP disfavors crystallization of the Gly-Ala-Gly-Ala-Gly-Ser repeats in SF due to steric factors. Hence, the solubility of the films formed from SF-GP conjugates can be modulated by the grafting density of the GP on the SF. This is very important for the development of degradable scaffolds for tissue engineering. Contact Angle Measurements. The surface hydrophilicity of the films was determined by measuring the water contact angle. The methanol treated films of SF, SF−GP25, and SF−GP50 were used to measure the contact angle. As shown in table 3, the unmodified SF has a contact angle of ∼67°, while the SF-GP25 and the SF-GP50 were found to have contact angles of ∼44° and ∼31°, respectively (Table 3). This implies that the attachment of GP causes dramatic increase in the hydrophilicity of the films, which increases with increase in the

Figure 5. Turbidity assay of SF-GP conjugates (A) SF−GP25, (B) SF− GP50, and (C) SF−GP125.

increases with increasing concentrations of SF−GP conjugates and finally reaches a plateau. At this point, the turbidity does not increase with increasing concentration of SF−GP

Table 2. Secondary Structure Content in Films Prepared from SF and SF−GP Conjugates secondary structure amide I film silk before methanol treatment silk after methanol treatment SF−GP25 SF−GP50 SF−GP125

amide II

amide III

random coil (1650 cm−1)

β-sheet (1630 cm−1)

random coil (1540 cm−1)

β-sheet (1520 cm−1)

random coil (1230 cm−1)

β-sheet (1270 cm−1)

+

−

+

−

+

−

+

+

+

+

+

+

+ + +

+ − −

+ + +

+ − −

+ + +

+ + −

3700

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Figure 6. Confocal microscope images of cast films of SF (a) and SF−GP25 (b) after immersion into FITC-Con-A.

conjugates and can be considered as an optimum value for the binding of the conjugate to Con-A. The optimum value for SF−GP25, SF−GP50, and SF−GP125 for [Con-A] = 0.5 mg/mL was found to be 22, 14, and 6.5 μg, respectively. This shows that the saturation for the SF−GP conjugates takes place in the order SF−GP25 < SF−GP50 < SF−GP125 (Figure 5). This is expected since amount of GP in the SF−GP conjugates increase in the order SF−GP125> SF−GP50> SF−GP25, and hence SF−GP125 is expected to bind at concentrations that are the lowest among the three SF−GP conjugates studied. Control experiments with silk-fibroin displayed no turbidity, showing that the specificity of poly(α-manno-O-lys)-functionalized SF (SF−GP25, SF−GP50, and SF−GP125) toward the lectin Con-A. Finally we wanted to probe whether the water-insoluble films formed from SF−GP25 were able to bind to the lectin Con-A. To prove this, we treated films formed from SF−GP25 (after methanol treatment) with fluorescently labeled Con-A and then imaged the films with confocal microscopy (Figure 6). Green fluorescence due to fluorescently labeled Con-A was observed from all over the SF-GP25 film, while in a control experiment performed similarly with unmodified SF film, no fluorescence was observed (Figure 6). The observation of fluorescence on the entire SF−GP25 film indicates that presence of mannose residues of the GP in the entire surface of SF−GP25. It also implies that the hydrophilic poly(α-manno-O-lys) GP chains present in SF−GP25 were exposed to the solvent and not buried in relatively more hydrophobic SF. This is extremely important for biological applications such as tissue engineering because the modified SF surface can be available for recognition with specific receptor proteins in aqueous medium. Cell Adhesion and Growth Assay. We then proceeded to preliminary experiments to determine whether silk films formed from SF−GP25 could support L6 growth. For these studies, coverslips were coated with 0.5% w/v solution of SF−GP25, dried, and treated with methanol to render the films insoluble in water. The L6 cells were then seeded on the SF−GP25 films and were imaged by epifluorescence microscopy after 24 h. It was observed that the L6 cells were able to attach and spread on the SF−GP25 films exhibiting a morphology that is typical of these cells. In order to determine whether the cell clustering observed was a result of cell death, we performed cell viability assay using a LIVE/DEAD assay and imaged the stained cells under a fluorescence microscope (Figure 7). In this assay, all the cells with green fluorescence indicates live cells (acridine orange stained), while the ones showing red fluorescence indicates dead cells (propidium iodide stained). Since most of the cells showed green fluorescence, it can be concluded that

Figure 7. Differential interference contrast (DIC) and LIVE/DEAD fluorescent images of L6 cells on SF−GP25 conjugate. Cells with green fluorescence indicates live cells.

silk film formed from SF−GP25 conjugate is biocompatible and supports the adhesion and growth of L6 cells. This is also indicative of the fact that there is no negative impact of using Cu(I) for the conjugation of GP to azide-labeled silk; the Cu removal protocol used by us was sufficient to remove all the copper from the conjugates (which is also supported by ICP studies).

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CONCLUSIONS We have for the first time synthesized an alkyne-terminated GP by ROP of α-manno-O-lys NCA with propargylamine as the initiator and then successfully conjugated it to azide-modified SF by CuAAC. By controlling the amount of GP grafted onto SF, we have made three SF−GP conjugates that differ in their ability assemble into films. SF−GP conjugates having a very high content of GP formed completely water-soluble brush-like polymer that displayed very high affinity toward the lectin ConA. Films cast from SF−GP conjugates using lower amounts of grafted GP were more stable in water, and the stability can be modulated by varying the amount of GP grafted. The water insoluble film SF−GP25 was also found to bind to Con-A as was seen by confocal microscopy. SF−GP conjugates are unique as they combine the structural properties of SF and the functional properties of the GPs. Preliminary experiments of cell growth and adhesion with rat skeletal muscle myoblast L6 cells show that the films formed from SF−GP25 are biocompatible and hence can have potential use in tissue engineering, drug 3701

dx.doi.org/10.1021/bm301170u | Biomacromolecules 2012, 13, 3695−3702

Biomacromolecules

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delivery, and gene delivery. Application of these SF−GP hybrids in liver tissue engineering by changing the pendant sugar moiety from mannose to galactose is underway in our laboratory.

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ASSOCIATED CONTENT

S Supporting Information *

Experimental procedures and spectral data for NCA monomers and GPs. FT-IR spectra of click reaction, 1H NMR spectra of all SF-GP conjugates. This information is available free of charge via Internet at http://pubs.acs.org/.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors acknowledge Dr. Ashish Lele for discussion on this manuscript, Dr. P Rajmohanan for NMR support, and Mr. J. Debgupta for contact angle measurements. S.D., D.P., and N.T. thank CSIR, New Delhi, for research fellowships. We thank CSRTI, Mysore for providing us with bivoltine Bombyx mori silk cocoons. We also thank DST, New Delhi for financial assistance (Indo-Korean project number INT/Korea/P-15).

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dx.doi.org/10.1021/bm301170u | Biomacromolecules 2012, 13, 3695−3702